Journal club presentation

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Raghavendra Institute of Pharmaceutical Education and Research - Autonomous
K.R.Palli Cross, Chiyyedu, Anantapuramu, A. P- 515721
Journal Club Presentation
Presented by
R. Jyothsna Naidu
19L81S0104
II year, I semester
M. Pharmacy
Department of Pharmacology

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Title, Author and Affiliations
Impact factor
4.732 (2020)
Impact factor: 4.732

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Literature
The above paper was chosen as it promotes my research study on the Neurobeneficial impact of probiotics on
gut dysbiosis
Literature search were done from Pubmed data base, Nature Scientific Reports, Elseiver
journals.
Journals were screened based on impact factor and
Science Citation Indexed (SCI), well peer reviewed.
Recent research articles from 2016-2019 were
screened
The following article is chosen after the above
justifications
Review articles were
excluded and research
articles were included

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Background- Gut Dysbiosis
• Gut microbiota consists of around 100 trillion microbial cells that provide
the host with a broad variety of metabolic functions.
• Human gut microbiome physiologically conducts variety of functions,
including digestion, the maintenance of immune and inflammatory
homeostasis and the nervous system through the microbiome-gut-brain
axis.
• Alteration in gut microbiome is Dysbiosis – cause for progression of
many pathologies of the disorder.
• A role for gut microbiota in the long-known link between diet and
emotion has emerged over the past few years, including evidence that
high-fat diet feeding promotes a “leaky gut” (i.e., increased intestinal
permeability) similar to the effect of chronic stress alone , and the
combination of high-fat diet and stress exposure may promote even
greater bacterial translocation.
• Increased circulating cytokines subsequent to bacterial translocation can
sensitize the HPA axis to stress-induced activation, and also can increase
anxiety- and depressive-like behaviours.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Background- Major Depressive
Disorder (MDD)
• Microbiome of depressed patients differs in terms of
composition and diversity
• Major depressive disorder (MDD) is a devastating yet
widespread chronic disorder
• Individuals suffering from MDD experience persistent low
mood, blunted or absent experience of pleasure, as well as a
wide range of cognitive, physical, and behavioral symptoms
that interfere with the individual’s capacity to function in
daily life.
• Causes of MDD- Diet, dysmetabolic conditions, activation of
HPA (hypothalamus–pituitary–adrenal) axis
• The gut-brain axis, this bidirectional communication network
- potential target for novel antidepressant treatments.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Background- Probiotics
• Probiotics are a combination of live beneficial
bacteria and/or yeasts that naturally live in your
body
• Probiotics are made up of good bacteria that helps
keep your body healthy and working well.
• Probiotic biotherapies are known to create a
healthy gut environment by balancing bacterial
populations and promoting their favorable
metabolic action.
• “Psychobiotics” - a live organ-ism that produces a
health benefit in patients suffering from a
psychiatric illness

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Aim and Objectives
Aim
To evaluate the effect of probiotic treatment on depression-related behaviour in rats on a control or high-fat diet
(HFD)
Objectives
• To induce High fat diet for the first five weeks.
 To feed the rats with probiotics and assess their protective effects independent of HFD
 To perform behavioral parameters for the HFD induced rats and estimate the protection of probiotics on
alterations in gut microbiome-brain axis caused by HFD.
 To perform plasma metabolomic analysis identify metabolites associated with probiotic treatment
• To examine mechanisms that may be involved in MDD - hippocampal and hypothalamic HPA axis
regulation, cytokine profile of stimulated immune cells and the complete plasma metabolomic profile.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Details of the Study
• Animals required: Forty male Sprague-Dawley rats
• Rat age : 4 weeks from birth
• Diet: control (CON) or high-fat diet (HFD) for ten weeks
• Treatment: multi-species probiotic formulation or vehicle for the last five weeks
• CON diet -11 kJ% (soybean oil), a protein content of 23 kJ% and a carbohydrate content of 66 kJ% (mainly corn starch)
• HFD diet- 60 kJ%(mainly beef tallow), a protein content of 20 kJ% and a carbohydrate content of 20 kJ% (maltodextrin and
sucrose).
• Probiotic formulation: probiotics consisted of eight bacterial strains (B. bifidum W23, B. lactis W52, L. acidophilus W37, L.
brevis W63, L. casei W56, L. salivarius W24, Lc. LactisW19, Lc. Lactis W58;
• Duration of the study: 16 weeks
• Diet- ad libitum with tap water
• Probiotics- 4.5 g(2.5 × 109CFU/g) of freeze-dried powder dissolved in 30 mL of tap water per cage.
All procedures complied with the EU Directive 2010/63/EU and with the Danish law regulating experiments on animals (permission
ID 2012-15-2934-00254).

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Study design and Outline

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Methods- Behavioural Parameters
• A test of hippocampus-dependent spatial memory which is often impaired in patients
suffering from MDD
• velocity (cm/s) was calculated as a measure of locomotor activity.
Barnes maze
• Commonly used screening tool for depressive like behavior in rodents
• Time spent on active behaviors (struggling and swimming) as well as immobility was calculated.
Forced swim test
• To assess locomotor activity
• distance moved by each animal (cm) was retrieved.
Open field test

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Metabolic Measures
Oral glucose
tolerance test
Plasma endotoxin
RNA extraction &
cDNA synthesis
Real-time qPCR Metabolomics
Measurement of
cytokines
Anti-CD3/28
stimulation of
PBMC

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Statistical analysis
• Significance level of α = 0.05 was used in all analyses. Group sizes were chosen on the basis of a
power calculation for FST( β = 0.2). Normality was assessed by QQ plots, and Bartlett’s test was used
to test for equal variances. Struggling time in FST and real-time qPCR data were power-transformed,
whereas LPS and cytokine levels as well as data from BM and OGTT were log-transformed before
analysis.
• Trek2 was analysed using Wilcoxon rank-sum test. Two-way ANOVA was used in analysis of body
weight, fasting glucose/insulin, FST, OF, LPS, BM recall trial and metabolomics with diet and
probiotic treatment as independent factors. Significant interactions were followed up by use of pair-
wise contrasts with Bonferroni-corrected p-values.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Statistical analysis
• Metabolomics data were also ana-lysed by use of a principal component analysis (PCA) to detect
major changes in analyte composition. In analysis of BM, OGTT and cytokines, a linear mixed
model analysis was conducted with rat identity as the random effect (intercept). Fixed effects
included day and session (BM), time (OGTT) or analyte (cytokines) and their interactions with diet,
treatment and diet-treatment. REML estimation was used, and residuals were considered independent
by trial (BM), time (glucose in OGTT) or analyte (cytokines).
• Contrasts of marginal linear predictions were used to test for differences between groups after the
models were built. False discovery rate (FDR) correction (Benjamini and Hochberg (Benjamini
andHochberg, 1995)) was applied to real-time qPCR data at a FDR level of 0.10. For metabolomics,
a less conservative FDR level of 0.20 was applied because of the hypothesis-generating purpose of
the analy-sis. All analyses were performed with Stata 14 (StataCorp LP, Texas,USA).

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Results
• Body weight & caloric intake
Body weights at study initiation were similar between groups. HFD led to a
marked increase in total caloric intake and in bodyweight at study end .
Probiotic treatment did not affect these measures.
• Forced swim test
Mean ± SEM. Probiotics markedly lowered depressive-like behaviour
(immobility) independently of diet.
Panel A depicts minute-wise immobility during the 7-min test trial, whereas
panel B shows total immobility duration in the test.
*** p < 0.001
P- main effect of probiotics.
CON: control diet; HFD: high-fat diet; VEH: vehicle; PRO: probiotics.
An antidepressant-like effect under both dietary conditions was further
confirmed by Bonferroni-adjusted post-hoc comparisons

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Results
• Open field test
No differences between groups were observed in distance
travelled during the 10-min open field session
• Barnes maze
1. Diet did, however, not affect distance travelled and no
effect of probiotic treatment was observed in initial trials.
2. In the recall-session 7 days later, HFD lowered the
distance travelled before locating the escape hole and
whereas a significant diet - treatment interaction was seen
for time to complete.
3. Here, post-hoc comparisons revealed that HFD led to
faster completion of the trial in VEH treated animals only.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Results
Oral glucose tolerance test
• Baseline fasting levels of blood glucose and plasma
insulin were not affected by diet or probiotic treatment,
although HFD strongly tended to increase insulin level
• A- During the OGTT, Mean ± SEM glucose levels rose
to a higher level in HFD rats than in CON rats
• B- Mean ± SEM HFD-fed rats also had higher insulin
levels that CON rats
• Probiotics-treated animals had lower glucose and insulin
levels during the test, but the improvements were not
statistically significant
• D- Diet did not interact with time course
* p < 0.05; ** p < 0.01

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Results
Cytokine production by PBMCs during 72 h of anti-CD3/28
stimulated cultures.
• A- Probiotic treatment affected the cytokine levels IL2, IL4
and IFNɣ (p < 0.05), Median and 25th/75th percentiles;
whiskers represent total range, diet had no such effect.
• B (relative distribution): In analysis of each cytokine in
percent of the total amount produced, an overall effect of
probiotics was observed again
Mean ± SEM . Probiotics increased the level of IL2, IL4 and
IFNɣ in percent of the total cytokine amount, but lowered IL6and
TNF .
Groups of similar treatment were merged to improve plainness.
(p) main effect of probiotics; *p < 0.05; **p < 0.01.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Results
Real-time qPCR
mRNA levels in hippocampus and hypothalamus; mean ± SEM, Data were analyzed with 2-way ANOVA
• HFD increased the transcript levels of the HPA axis regulating genes Mr, Crh-r1, Crh-r2 , 11β-hsd1
• Probiotics lowered the expression of these genes genes Mr, Crh-r1, Crh-r2, (exception of 11β-hsd1)
• The hippocampal expression of Bdnf, Trek2 and Traak, which are related to structural plasticity and neuroprotection, was lowered by
HFD.
• Probiotics oppositely increased the level of Trek2, and Traak.
• In hypothalamus, only one change was observed namely, HFD increased the level of Crh-r2
• Two significant diet - treatment interactions did not pass the FDR correction (Traak in hippocampus and Pomc in hypothalamus ).
P- Significant effect of probiotic treatment
D- significant effect of diet.
No diet - treatment interactions were observed.
.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Results
Metabolomics
Mean ± SEM, Data were analyzed with 2-way ANOVA
• 455 plasma metabolites were detected in at least 30 animals
• Thirteen metabolites were found to be affected by probiotic treatment, and three of these changes remained significantly different after
FDR( false discovery rate) correction
• Microbial tryptophan metabolites, metabolite related to amino acid metabolism; namely, acetylornithine - upregulated by Probiotics
• HFD affected the level of 209 metabolites (data not presented)- lower plasma trypto-phan level and higher levels of the tryptophan
catabolite quinolinic acid.
• No diet- treatment interactions were found.
P- Significant effect of probiotics after FDR correction
D-Significant effect of diet after FDR correction

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Results
Plasma endotoxin
• HFD- increased plasma LPS (Lipopolysacharide) level
(p = 0.005)
• Probiotics- didn’t affect the concentration (p = 0.5)
• D- main effect of diet.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Interpretation
• High-fat diet led to a clear metabolic stress as indicated by increased body weight, caloric intake,
glucose and insulin levels
• HFD did not affect depressive-like behavior (forced swim test)
• It was found that HFD improved memory in the Barnes maze, the diet-induced improvement was,
however, only seen on day 4 and in the recall trial.
Plasma endotoxin:
• Previous studies- LPS has been suggested to activate especially monocytes and induce depressive-
like symptoms
• HFD- increased plasma LPS in present study
• Probiotics- yet no change in LPS (lipopolysaccharide)

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Interpretation
Measurement of cytokines:
Probiotic treatment- Increase in T lymphocyte-derived cytokines IL2, IL4 and IFNγ and decrease in TNFα and
IL6
• In several metanalyses, macrophage-derived plasma cytokines, including TNFα and IL6, are consistently
found to be modestly increased in depressed patients.
• On the other hand a meta-analysis concluded that T cell activity is generally compromised in MDD.
• T cell subsets have recently been found to support the structural plasticity of the CNS-normal brain
functioning of mice
MDD may therefore be associated with an overactivity of the innate arm (cytokines, interleukins) of the immune
system to the detriment of the adaptive arm (T- lymphocytes)
• This Interestingly present cytokine findings may reflect that probiotics shifted the balance towards the
adaptive arm.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Interpretation
RT-qPCR
• Probiotic treatment- altered the expression of several hippocampal genes related to HPA axis feedback
Clearly,
• Hippocampal transcript levels of Crh-r1 and Crh-r2- by Probiotics.
• CRH-R1 signaling - in stress and depression and receptor antagonists were shown to possess
antidepressant like effect in animals.
• HFD also changed the expression of several genes in hippocampus and also increased the mRNA level of
several factors involved in HPA axis regulation.
• HFD and probiotics had oppositely directed effects on all affected genes.
• HFD- Bdnf, Traak and Trek2 mRNA levels in animals
• Traak and Trek2- Protection against glutamatergic excitotoxicity that has been associated with depression

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Interpretation
Metabolomics:
• HFD- gut microbiota diversity in mice, generally possess fatty acids (antimicrobial effects)
• Plasma metabolomic profile- not much affected by probiotic treatment
• HFD- markedly affected the plasma metabolome
• HFD - plasma quinolinic acid (increased need for NAD for fatty acid metabolism)
• Tryptophan and its metabolites, including kynurenine pathway metabolites, are believed to play an important role in
MDD,
• Probiotics- increased the level of two microbial tryptophan metabolites
IPA (Indole Propionic acid) -neuroprotective properties and restrain CNS inflammation
IPA could potentially mediate some of the immunomodulatory effects of the probiotics
4- EthylPhenylSulphate (EPS)- by probiotics although the change did not pass FDR correction (False Discovery Rate)
In mice EPS responsible for aberrant behaviors with an autism like phenotype which was similarly reduced by probiotic
treatment.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Authors conclusion
The multi-species probiotics used in the present study clearly lowered depressive-
like behavior, and HFD did not affect the effectiveness of the treatment. The latter
point is of great importance given the high metabolic comorbidity in depression, and
it further strengthens the generalisability of our findings. On the molecular level,
probiotics were shown to interact with pathophysiological mechanisms believed to
play an important role in MDD, including the immune system, hippocampal HPA
axis regulation and microbial tryptophan metabolism. Our study therefore confirms
probiotics as a promising candidate for the treatment of depression. Now we just
need the clinical trials.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Critical Analysis
Title: Probiotic treatment reduces depressive-like behavior in rats independently of diet
The title was justified and the provided results in the study have shown anti depressive effect when administered with
Probiotics.
Background of research:
Well structured background on Gut microbiota, HPA axis, HFD consumption may lead to increased basal and stress-
induced plasma corticosterone levels, IPA , (TLR4) signaling which linked to present study.
Aim and objectives:
All the objectives mentioned were evaluated and and proper interpretation of data with statistical analysis were provided
Validity of methods
All validated methods were used for behavioral parameters and all the manufactures instructions were followed for the
metabolic measurements like metabolomic profiling and RT-qPCR.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Critical Analysis
Animals and diet
All procedures complied with the EU Directive 2010/63/EU and with the Danish law regulating exper-iments on animals
(permission ID 2012-15-2934-00254).
Treatment:
Appropriate critical guidelines were followed
Results
Results of each parameter were interpreted with respective statistical analysis
Conclusion
Conclusions were valid and supported the present study.
Author limitations:
• Young adult rats were included, gut microbiota changes with age and the effect of probiotics may thus be different
in older rats.
• The effect of probiotics was measured in SD rats and not in a depression-related disease model.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Strengths
• Probiotic treatment markedly reduced the level of depressive-like behavior independently of diet.
• Identified corresponding changes in three physiological systems that could potentially mediate the observed
antidepressant-like effect.
• Firstly, pronounced shifts in the pattern of produced cytokines were observed.
• Secondly, changes in the hippocampal expression of genes related to HPA axis regulation were evident; and
• finally, the concentration of a number of plasma metabolites were altered.
• previous studies has utilized treatment with only one or two bacterial strains, we used a multi-species composition of
eight different strains. this notion may partially explain the unequivocally antidepressant-like effect in non-stressed
rats.
• Present finding HFD did not interact with the antidepressant-like effect of probiotics only further expands the
potential group of amenable patients
• Results therefore urge for intensified research into these metabolites and their potential role in MDD.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Overview
• The gut microbiota has recently emerged as an important regulator of brain physiology and behavior in animals, and ingestion of certain bacteria
(probiotics) therefore appear to be a potential treatment for major depressive disorder (MDD).
• Study aimed at investigating whether the habitual diet may interact with the effect of pro-biotics on depression-related behavior and further examined
some potentially involved mechanisms underlying the microbe-mediated behavioral effects.
• Forty male Sprague-Dawley rats were fed a control (CON) or high-fat diet (HFD) for ten weeks and treated with either a multi-species probiotic
formulation or vehicle for the last five weeks.
• Independently of diet, probiotic treatment markedly reduced depressive-like behavior in the forced swim test , probiotic treatment skewed the cytokine
production by stimulated blood mononuclear cells towards IFN , IL2 and IL4 at the expense of TNF and IL6.
• In addition, probiotics lowered hippocampal transcript levels of factors involved in HPA axis regulation (Crh-r1, Crh-r2 and Mr), whereas HFD
increased these levels.
• A non-targeted plasma metabolomics analysis revealed that probiotics raised the level of indole-3-propionic acid, a potential neuroprotective agent.
• findings clearly support probiotics as a potential treatment strategy in MDD. Mechanistically, the HPA axis, immune system and microbial tryptophan
metabolism could be important in this context.
• Although these marked diet-induced changes in gene expressions were not directly mirrored in the level of depressive-like behavior, it is still likely that
animals on HFD are more susceptible to episodes of intercurrent stress.

RIPER
AUTONOMOUS
NAAC &
NBA (UG)
SIRO- DSIR
Applicability of findings of article
• The concept of psychobiotics, bacteria with positive effects on mental health
• To develop therapeutic strategies for treating stress-related disorders based on relationship
between diet and the microbiota-gut-brain axis
• To fill the gap between laboratory scientists studying the gut-brain connection and people
with psychiatric disorders and cognitive challenges
• These results emphasize the complex influence of dietary factors on stress related
outcomes, and highlight the need for additional research to unravel complex interactions
and causal relationships among diet, stress exposure, and the microbial gut-brain axis.

Journal club presentation

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Journal club presentation

Similar to Journal club presentation (20)

More from Ravilla Jyothsna Naidu

More from Ravilla Jyothsna Naidu (7)

Recently uploaded

Recently uploaded (20)

Journal club presentation